The role of BST4 in the pyrenoid of Chlamydomonas reinhardtii

The eukaryotic algal CO2-concentrating mechanism (CCM) is based on a Rubisco-rich organelle called the pyrenoid, which is typically traversed by a network of thylakoid membranes. BST4 is a bestrophin-like transmembrane protein that has previously been identified in the model alga Chlamydomonas reinhardtii as a putative tether that could link the traversing thylakoid membrane network to the Rubisco matrix. In the present study, we show that BST4 forms a higher order complex assembly that localizes to the thylakoid network within the pyrenoid. However, investigation of a bst4 knock-out mutant in Chlamydomonas showed that the absence of BST4 did not result in a CCM-deficient phenotype and that BST4 is not necessary for the formation of the trans-pyrenoid thylakoids. Furthermore, heterologous expression of BST4 was not sufficient to facilitate the incorporation of thylakoids into a reconstituted Rubisco condensate in the land plant Arabidopsis. Subsequent analyses revealed that bst4 was under oxidative stress and showed enhanced non-photochemical quenching associated with CO2 limitation and over acidification of the thylakoid lumen. We conclude that the primary role of BST4 is not as a tethering protein, but rather as an ion channel involved in pH regulation in pyrenoid-based CCMs.

真核藻类的二氧化碳浓缩机制（CCM）基于一种富含核酮糖 - 1,5 - 二磷酸羧化酶/加氧酶（Rubisco）的细胞器，称为淀粉核，它通常被类囊体膜网络贯穿。BST4是一种类Bestrophin的跨膜蛋白，先前在模式藻类莱茵衣藻中被鉴定为一种可能的连接物，它可以将贯穿的类囊体膜网络与Rubisco基质连接起来。在本研究中，我们表明BST4形成一种高阶复合组件，定位于淀粉核内的类囊体网络。然而，对莱茵衣藻中bst4敲除突变体的研究表明，BST4的缺失不会导致CCM缺陷表型，并且BST4对于跨淀粉核类囊体的形成不是必需的。此外，BST4的异源表达不足以促进类囊体整合到陆生植物拟南芥中重构的Rubisco凝聚物中。后续分析显示，bst4处于氧化应激状态，并显示出与二氧化碳限制和类囊体腔过度酸化相关的非光化学猝灭增强。我们得出结论，BST4的主要作用不是作为一种连接蛋白，而是作为一种参与基于淀粉核的CCM中pH调节的离子通道。